Epidemiological studies show that the transfusion of older stored blood is associated with increases in mortality, serious infections, multi-organ failure, and hospital length of stay. Our research is based on the overarching hypothesis that the adverse effects of the transfusion of stored blood result from the acute delivery of hemoglobin iron to the monocyte-macrophage system. By current FDA standards, a unit of stored red blood cells (RBC) is clinically acceptable for transfusion if as much as 25% of the RBC is cleared within 24 hours, thereby delivering a substantial dose of iron to the monocyte-macrophage system. We hypothesize that the pro-oxidant effects exerted by acute exposure to these massive amounts of hemoglobin iron induce a pro- inflammatory cytokine response that may lead to the serious consequences seen in human studies. Our pre- clinical studies using a well-characterized mouse model demonstrate that the transfusion of older stored RBC acutely delivers hemoglobin iron to the spleen, kidney, and liver, induces an intense pro-inflammatory cytokine response, and synergizes with the effects of endotoxin (a standard sepsis model) to enhance and prolong cytokine storm. Therefore, the main goal of the current proposal is to establish the relevance of these novel pre-clinical observations in humans. To this end, we propose carefully-controlled, prospective studies of healthy volunteers and patients with hemoglobinopathies. We will first study healthy individuals to avoid the confounding factors present in prior studies of critically-ill or surgical patients that examined whether transfusions of older stored RBC produce adverse effects. We will then extend these findings to two relevant human disease settings, by studying stable patients with either sickle cell disease or beta-thalassemia who regularly receive simple transfusions. Because these patients are treated with chronic iron-chelating therapy, which can be safely discontinued for short periods of time, this will allow us to evaluate our novel hypothesis implicating the role of acute iron delivery. Aim #1 will test the hypothesis that transfusion of older stored RBC into healthy human volunteers induces an acute pro-inflammatory cytokine response. Aim #2 will test the hypothesis that transfusion of older stored RBC induces an acute pro-inflammatory response in chronically transfused patients with either sickle cell disease or beta-thalassemia. In addition, we will determine whether other standard RBC products often used in this setting (i.e. washed RBC and cryopreserved RBC) induce similar effects. Finally, Aim #3 will test the hypothesis that treating sickle cell disease and beta-thalassemia patients with iron chelators will prevent the acute pro- inflammatory response induced by transfusing older stored RBC. The insights gained from completing this proposal will have an immediate impact on the current practice of transfusion medicine and will provide the foundation for developing rationally-designed approaches to improve the quality of human donor RBC and of human transfusion therapy.